RESTRICTION ISOTYPING OF COMMON APO E ISOFORMS ON AGOROSE GEL
2012
SUMMARY Apolipoprotein E (apo E) is the main protein constituent of very low density lipoproteins (VLDL) and chylomicrons, that serves as a ligand for B/E and E receptors. There are three common apo E isoforms that differ at positions 112 and 158, and are called E2, E3, and E4. The isoform of apo E is an important determinant of plasma lipid levels and relative risk of atherosclerosis. In this study we present a simple and rapid method for routine analysis of apo E isoforms. We have used restriction isotyping for the detection of the common apo E isoforms. Genomi DNA was amplified with specific primers that included the polymorphic regions of amino acids 112 and 158. The amplification products were digested with the restriction enzyme Cfo I. The digested products were separated on NuSieve agarose-agarose gel electrophoresis, instead of polyacrylamide gel. Of the eighteen samples studied, five different apo E genotypes were identified. There is potential neurotoxicity and difficulty of polyacrylamide gel electrophoresis. Use of NuSieve agarose-agarose instead of polyacrylamide for apo E genotyping is easier, less time-consuming and more favorable for routine analysis. Key Words: Apolipoprotein E, restriction isotyping, Cfo I, NuSieve agarose, polyacrylamide OZET SIK GORULEN APO E IZOFORMLARININ AGAROZ JELDE RESTRIKSIYON TIPLEME YONTEMIYLE GOSTERILMESI Apolipoprotein E cok dusuk dansiteli lipoprotein ve silomikronlarin temel protein icerigini olusturmaktadir ve B/E ve E reseptorlerine baglanmakta ligand olarak fonksiyon gormektedir. Amino asid dizisinin 112 ve 158. pozisyonlarinda farklilik gosteren uc degisik apo E izoformu, E2, E3 ve E4 olarak isimlendirilmektedir. Apo E izoformu plazma lipid duzeyinin ve ateroskleroz riskinin belirlenmesinde onemlidir. Bu calismada apo E izoformlarmin gosterilmesinde rutinde yararlanilabilecek basit ve hizli bir metod kullanilmistir. Sik gorulen apo E izoformlarmin saptanmasinda restriksiyon tipleme yontemi kullanilmistir. Genomik DNA spesifik primerler kullanilarak amplifiye edilmistir. Amplifikasyon urunu Cfo I restriksiyon enzimi ile kesilerek, ortaya cikan DNA fragmanlari poliakrilamid jel yerine NuSieve agaroz-agaroz jel elektroforezinde ayristirilmistir. Calisilan onsekiz ornekte bes farkli apo E genotipi saptanmistir. Poliakrilamid norotoksik bir madde olup, jel elektroforezi zordur. Apo E genotip belirlemesinde poliakrilamid yerine NuSieve agaroz-agaroz karisiminin kullanimi daha kolay olup, daha az zaman gerektirmektedir. Ayrica rutin analize daha uygun gorunmektedir. Anahtar Kelimeler: Apolipoprotein E, restriksiyon tipleme, Cfo I, NuSieve agaroz, poiakrilamid
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